(1) To develop animal disease (end host) and persistence (reservoir host) models: Over the past years we have developed and characterized rodent and nonhuman primate disease models for infections with influenza A viruses, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), henipaviruses (Nipah and Hendra), and hantaviruses causing Hantavirus Pulmonary Syndrome (HPS). The two nonhuman primate MERS models, rhesus macaque and common marmoset, were further refined with a comparative pathology study. The results suggested that increased virus replication and the local immune response to MERS-CoV infection play a role in the severity of pulmonary pathology. We also investigated whether domestic pigs could serve as an amplifying/intermediate species for MERS-CoV or as a disease model. Pigs were inoculated intranasally and intratracheally with a high dose of MERS-CoV but did not develop signs of disease nor lesions in the respiratory tract. They are unlikely to serve as an amplifying/intermediate species for MERS-CoV. We have also further characterized the rhesus macaque HPS model to investigate mechanisms of disease pathogenesis. We are currently studying specific inflammatory events to gain understanding of what contributes to disease as well as to develop a therapeutic. Much of our work on hantaviruses in the past year was aimed at determining how the natural reservoirs can support elevated levels of virus replication without disease. We have previously shown that Sin Nombre virus elicits an initial inflammatory response in deer mice, the natural reservoir, but this response turned into an active anti-inflammatory response, as indicated by the activity of virus-specific T regulatory cells. Currently we are determining the requirement of these T regulatory cells to the suppression of the anti-inflammatory response by using T cell depletion strategies. (studies ongoing) (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: Severe influenza virus infections are often associated with bacterial co-infections. To study a potentiating effect of co-infection we performed a study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 showed enhanced clinical disease. Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement. Studies to decipher the mechanisms behind these observations were objectives over the past year and are still ongoing. (studies ongoing) We could identify the early target cells of Nipah virus infection in the hamster disease model. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination. This has important implications for the development of vaccine and therapeutics/antivirals. We could show that for hantaviruses adaptation to cell culture leads to loss of virulence. Therefore, we have established colonies with different mouse species (Peromyscus maniculatus; Apodemus flavicollis) for studying virus-reservoir interaction. These colonies will also be used to produce stock virus for in vivo work. (studies ongoing) (3) To identify and characterize host responses to viral infection to develop therapeutics: In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We have now shown that pre-exposure treatment reduces clinical signs of disease and viral titers in target organs. (studies ongoing) We have also tested efficacy of three monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset. These mAb had shown efficacy in mouse models of MERS-CoV infection. Unfortunately, none of the mABs showed significant reduction in disease burden and viral lung load in the nonhuman primate model suggesting that treatment with mABs may likely not very efficacious. Confirmatory studies and treatment with mAB cocktails are either ongoing or planned. We have tested the therapeutic efficacy of alisporivir, a non-immunosuppressive cyclosporin A-analog, against MERS-CoV and SARS-CoV. Low-micromolar concentrations of alisporivir inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. We have tested the efficacy of the antiviral compound GS-5734 against MERS-CoV in the rhesus macaque model. Pre-exposure treatment resulted in reduction of disease burden and viral lung loads. In contrast, post-exposure treatment with GS-5734 showed only minor effects. Confirmatory studies in the marmoset model are planned. (studies ongoing) (4) To develop protective vaccines: We continued with our efforts to develop a universal vaccine against influenza A viruses. We currently are applying two approaches: i) expression of highly conserved B cell epitopes from two separate helical regions within the hemagglutinin stalk that have shown to afford heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region to increase antibody responses against otherwise poorly antigenic epitopes. We used the Cytomegalovirus (CMV) vector platform for these studies, which can induce long-lasting immune responses (both T cell and antibody). Unfortunately, first attempts using the mouse model of influenza A viruses were rather discouraging. We will continue to optimize the CMV platform but have also started to use the vesicular stomatitis virus (VSV) as an alternative platform. (studies are ongoing) For MERS, we have obtained very promising results with a DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species, mice, macaques and camels using a prime/boost/boost approach. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. Recently, we were successful in shortening the vaccination strategy for potential application of this vaccination approach in emergency situations to prevent MERS-CoV infection. (manuscript in preparation) To generate a vaccine against Nipah virus infection, we used the VSV platform to express single Nipah virus glycoproteins (G or F) as the immunogens. The vaccines elicited strong antibody responses in hamsters and nonhuman primates and protected them from lethal Nipah virus challenge. We could demonstrate that the vaccines elucidated strong neutralizing responses and primed the CD8+ T cell responses. To investigate the limits of the efficacy of this vaccine, we used the hamster model and showed that this vaccine still provided partial protection when administered on the day of Nipah virus challenge. The VSV vaccine vectors expressing the Nipah virus G protein is currently scheduled for GMP production.